The Gram-positive bacterium Romboutsia ilealis harbors a polysaccharide synthase that can produce (1,3;1,4)-β-d-glucans

(1,3;1,4)-β-d-Glucans are widely distributed in the cell walls of grasses (family Poaceae) and closely related families, as well as some other vascular plants. Additionally, they have been found in other organisms, including fungi, lichens, brown algae, charophycean green algae, and the bacterium Sinorhizobium meliloti. Only three members of the Cellulose Synthase-Like (CSL) genes in the families CSLF, CSLH, and CSLJ are implicated in (1,3;1,4)-β-d-glucan biosynthesis in grasses. Little is known about the enzymes responsible for synthesizing (1,3;1,4)-β-d-glucans outside the grasses. In the present study, we report the presence of (1,3;1,4)-β-d-glucans in the exopolysaccharides of the Gram-positive bacterium Romboutsia ilealis CRIBT. We also report that RiGT2 is the candidate gene of R. ilealis that encodes (1,3;1,4)-β-d-glucan synthase. RiGT2 has conserved glycosyltransferase family 2 (GT2) motifs, including D, D, D, QXXRW, and a C-terminal PilZ domain that resembles the C-terminal domain of bacteria cellulose synthase, BcsA. Using a direct gain-of-function approach, we insert RiGT2 into Saccharomyces cerevisiae, and (1,3;1,4)-β-d-glucans are produced with structures similar to those of the (1,3;1,4)-β-d-glucans of the lichen Cetraria islandica. Phylogenetic analysis reveals that putative (1,3;1,4)-β-d-glucan synthase candidate genes in several other bacterial species support the finding of (1,3;1,4)-β-d-glucans in these species.


Statistics
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March 2021
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The experiment was performed in triplicates and the data were analyzed descriptively.
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All experimental measurements were performed in triplicate, except for sugar linkage analysis which was done in duplicate. Error bars represent SD. For the confocal and electron microscopies, at least three slides/grids were prepared independently. All attempts of replication were successful and gave similar results.
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